Biology subjects

Ferreira, A.

Publications and source records attributed to Ferreira, A..

At least 19 recordsLinked to original sources

bioRxiv2026

NeoDBS: Open-Source Platform for Visualization and Analysis of Electrophysiological Recordings from Deep Brain Stimulation Systems

Optimization of deep brain stimulation (DBS) therapy for neurological and neuropsychiatric disorders depends on objective quantitative biomarkers that can guide stimulation parameter adjustments. With the recent introduction of new-generation DBS systems capable of simultaneously stimulating brain activity and recording local field potentials (LFP), there is increasing demand for platforms that enable efficient visualization and analysis of these signals for electrophysiological biomarkers identification. To address the limitations of currently available toolboxes that require advanced signal processing skills and rely on proprietary software, we present NeoDBS, an open-source Python platform designed for ingestion and advance signal visualization and processing of LFP signals from DBS systems through an easy-to-use graphical interface. NeoDBS is a user-centered platform that offers predefined analysis pipelines with the aim of facilitating electrophysiological biomarker investigation for DBS across different brain disorders. Custom analysis pipelines are also available for users to leverage the signal analysis tools to their research needs. Critical functionalities for longitudinal biomarker research are featured in NeoDBS, such as batch file processing and event-locked analysis for in-clinic and at-home recordings. This combination of accessibility, user-experience and advanced signal processing tools makes NeoDBS an environment that propels easy and fast electrophysiological biomarker research for DBS, across patients, sessions, and stimulation parameters.

bioengineering↗

bioRxiv2026

No evidence of sentinel behaviour in a highly social bird based on an artificial set-up

Sentinel behaviour occurs when individuals use raised positions to scan for predators while the rest of the group forages. Here, we investigated whether a colonial cooperatively breeding species that forages in large groups, the sociable weaver, Philetairus socius, displays sentinel behaviour. This behaviour has been reported in species with similar ecology, behaviour and foraging habits, (e.g. ground foraging in open habitats where aerial predators are common) and, hence, we expected that it could occur in sociable weavers. On the other hand, sentinel behaviour appears to be less common in species that live in very large groups. We used an experimental set-up consisting of an artificial feeding station and perches to assess occurrence of sentinel related behaviours: (i) perching events > 30s on an elevated position, (ii) head-movements and (iii) alarm calling. Birds were seldom observed perching while others fed, and those that did, perched for periods that were too short to be considered as sentinel behaviour (less than 5s on average). Our results suggest that this behaviour is uncommon or even absent in sociable weavers. We discuss whether other factors such as foraging in very large groups, or interspecific foraging associations might make sentinel behaviour less important in this species.

animal behavior and cognition↗

bioRxiv2026

Sex-specific Flexibility in Breeding and Helping Strategies in the Sociable Weaver (Philetairus socius)

Helping-at-the-nest is often viewed as a precursor to reproduction, but switching between breeder and helper roles has been documented in some species. Such flexibility should depend on the opportunities and benefits of helping, which could differ between sexes due to dispersal strategies and social structure. However, whether breeding-helping flexibility is widespread and sex-specific remains unknown. Here, we investigated sex-specific strategies in breeding-helping flexibility using a 10-year dataset (1955 individuals) on sociable weavers (Philetairus socius), a colonial cooperative breeder in which males are typically philopatric whereas females disperse. Both sexes helped for several years, with males helping more frequently than females and for twice as long (0-13 vs 0-10 years). Around 40% of non-dispersing birds never became breeders and 50% of individuals bred without helping first. Both sexes switched roles between- and within-seasons but males were more likely to alternate (respectively four and six times more than females and up 15 switches across seven years). Our study reveals important flexibility and sex differences in breeder-helper roles, consistent with sex-biased dispersal, spatial breeding proximity and possible indirect fitness benefits. These factors could play a role promoting the evolution of helping across life-stages, not only pre-reproduction, but also reproductive and post-reproductive individuals.

evolutionary biology↗

bioRxiv2025

Experimentally assessing the role of foraging encounters on social partner choice in a kin-biased bird society

Kin-based social environments are often characterized by prolonged parent-offspring associations and limited dispersal in at least one sex, leading to groups composed of kin as social partners. These high rates of social interactions among relatives in cooperative societies make it difficult to distinguish whether social partner choice is driven by kinship or by repeated encounters with individuals situated at proximity. Here, we experimentally increased the number of foraging encounters among non-kin at a custom feeder setup in the sociable weaver (Philetairus socius), a colonial, cooperatively breeding bird with a kin-based social structure. Individuals encouraged to forage more frequently with non-relatives subsequently developed stronger foraging associations with non-kin at separate, unrestricted feeders. These findings show that repeated opportunities for interaction can promote non-kin social associations even within a kin-based society, contributing to the understanding of proximate mechanisms that underlie social structure.

animal behavior and cognition↗

bioRxiv2025

Lipid droplets in felid kidneys: prevalence and composition by lipidomics

An accepted and common phenotypic curiosity of Felidae is the presence of intracytoplasmic lipid droplets in renal proximal tubule epithelial cells (RPTEC), also frequently in urine (lipuria). Both outcomes are currently considered, and taught, as incidental - without obvious pathophysiological consequence. This contrasts markedly with clinical (human) medicine where lipid vacuoles in RPTEC are usually associated with metabolic or chronic disease, such as CKD. Despite domestic felids having a high incidence of CKD as they age, no study has fully characterised feline RPTEC lipid droplets in the context of CKD. Here, we first characterised the incidence of RPTEC lipid in domestic cat (with/without CKD or chronic interstitial nephritis) versus domestic dog and Scottish Wildcat, across a wide age range. Felids (domestic, wildcat) consistently had greater renal lipid content than dogs at all ages studied. Intracytoplasmic lipid extraction with chromatography, fatty acid characterisation and mass spectrometry-based lipidomics revealed unusual presence of a panoply of novel lipids found only in domestic cat: lipids were primarily modified (i.e. less polar) ether-soluble triacylglycerols including monoalkyldiacylglycerols (MADAGs) and other branched-chain fatty acids. We suggest common presence of such rare lipid species in tubular lipid droplets in domestic cat reflects an aspect of felid biology that parallels age-related disease prevalence, in particular, being associated with the aetiopathogenesis of chronic renal interstitial nephritis (CIN) - a hallmark of CKD in felids.

zoology↗

bioRxiv2025

Immune responses to human pathogens exposed to simulated Mars conditions

The identification of health risks associated with long-term crewed missions to Mars is critical for mission planning and crew safety. Human-associated pathogens can be part of the microbiome and are likely to be transported during these missions. This study examines the immunological responses of human immune cells stimulated with non-fastidious bacterial species that cause opportunistic infections, i.e. Klebsiella pneumoniae and Serratia marcescens, after exposure to simulated Martian conditions, including UV radiation, desiccation and atmospheric pressure. We observed that exposure of the bacteria to these conditions altered cytokine secretion, reactive oxygen species (ROS) production, and phagocytic activity in human peripheral blood mononuclear cells (PBMCs). Specifically, exposure to desiccation reduced cytokines and ROS production, indicating impaired innate immune recognition and stimulation. Notably, the altered immune response was partially restored when desiccated bacteria were regrown in standard media. Flow cytometry revealed decreased bacterial size and complexity of both species post-exposure. These findings indicate that Martian conditions induce bacterial morphological and physiological changes, which could impair immune recognition and response. Expanding these studies to in vivo models and a broader range of potentially pathogenic microorganisms is essential to estimate infection risks during Mars missions, which is vital for developing strategies to mitigate infection risks and maintain astronaut health during long-term space travel. ImportanceSince Yuri Gagarins 1961 flight, human space exploration has expanded, unintentionally transporting microorganisms, including pathogens, into space environments. Our previous studies demonstrated that opportunistic pathogens like Klebsiella pneumoniae and Serratia marcescens can survive simulated Mars conditions. With upcoming Mars missions, it is crucial to understand how such conditions influence these pathogens and their interaction with the human immune system. This research evaluates immune responses to bacteria pathogens exposed to Martian stressors such as UV radiation and desiccation, revealing significant changes of the immune responses to the exposed bacteria. These findings provide essential insights into the health risks that astronauts may face if infected with Mars-adapted pathogens. Understanding these interactions will help to develop preventive strategies and therapeutic measures, ensuring the safety and health of crew members during long-term missions. Ultimately, this work contributes to the broader objective of safe human exploration and colonization of Mars.

immunology↗

bioRxiv2024

Moving towards more holistic validation of machine learning-based approaches in ecology and evolution

Machine-learning (ML) is revolutionizing field and laboratory studies of animals. However, a challenge when deploying ML for classification tasks is ensuring the models are reliable. Currently, we evaluate models using performance metrics (e.g., precision, recall, F1), but these can overlook the ultimate aim, which is not the outputs themselves (e.g. detected species or individual identities, or behaviour) but their incorporation for hypothesis testing. As improving performance metrics has diminishing returns, particularly when data are inherently noisy (as human-labelled, animal-based data often are), researchers are faced with the conundrum of investing more time in maximising metrics versus doing the actual research. This raises the question: how much noise can we accept in ML models? Here, we start by describing an under-reported factor that can cause metrics to underestimate model performance. Specifically, ambiguity between categories or mistakes in labelling validation data produces hard ceilings that limit performance metrics. This likely widespread issue means that many models could be performing better than their metrics suggest. Next, we argue and show that imperfect models (e.g. low F1 scores) can still be useable. Using a case study on ML-identified behaviour from vulturine guineafowl accelerometer data, we first propose a simulation framework to evaluate robustness of hypothesis testing using models that make classification errors. Second, we show how to determine the utility of a model by supplementing existing performance metrics with biological validations. This involves applying ML models to unlabelled data and using the models outputs to test hypotheses for which we can anticipate the outcome. Together, we show that effects sizes and expected biological patterns can be detected even when performance metrics are relatively low (e.g., F1: 60-70%). In doing so, we provide a roadmap for validation approaches of ML classification models tailored to research in animal behaviour, and other fields with noisy, biological data. HighlightsEvaluating machine learning (ML) models must go beyond performance metrics Mislabels in validation data leads to underestimation of models performance Underestimated metrics can cause research delays despite models being useful We propose simulations and biological validations to evaluate model performance Models with low standard metrics can still be powerful for hypothesis testing

ecology↗

bioRxiv2024

Growth compensation upon changes in tissue size in the Drosophila abdomen

Attaining the appropriate size during development is essential for the function of animal tissues and organs. Robust tissue size control implies the existence of compensatory mechanisms that allow developing systems to recover from growth perturbations. However, the difficulty of directly observing normal or compensatory developmental growth means we have little understanding of the cellular behaviours that confer robustness to tissue size control. Here, we study how growth perturbations affect proliferation kinetics and the timing of growth termination of Drosophila histoblasts, the progenitors that give rise to the adult abdominal epidermis. Histoblasts undergo extensive growth and proliferation during the pupal stage, which is accessible for long-term live-imaging and precise quantitative analysis. By manipulating cell number or volume prior to the pupal growth phase, we changed the starting size of the abdomen primordium, then observed how the histoblasts adapted to these changes by altering their growth dynamics. We show that, upon a decrease in starting tissue size, the histoblasts compensate by extending their temporal proliferative window, undergoing additional cell cycles, as well as increasing their apical area to maximise coverage of the abdominal surface. When initial tissue size is increased, the histoblasts undergo fewer division cycles and arrest proliferation earlier than normal. Thus, the proliferative window of this tissue is flexible enough to buffer for changes in tissue size. Our data also suggest that the histoblasts sense both spatial and temporal cues to arrest their growth at the appropriate time and ensure accurate tissue size control.

developmental biology↗

bioRxiv2024

Growth characterization and biostimulant potential of Coelastrella sp. D14 a greenmicroalga isolated from a solar panel.

Extremophile environments are an important source for finding microorganisms with a potential for biotechnological applications. Among these microorganisms, microalgae contribute to several sustainable processes such as wastewater treatments or nutrition. In this work it was characterized a microalga isolated from a solar panel. The morphological and phylogenetic analysis revealed that the isolate collected was a Coelastrella strain. Cultivation and stress experiments has shown that Coelastrella sp. D14 can resist a long period of desiccation and it can grow on cheap sources such as piggery wastewaters (PWW). This work reports that a Coelastrella strain displays biostimulant properties with a germination index of 123% on Lepidium sativum when D14 biomass grown at 10% piggery effluent was used. Altogether, these results suggest that this novel strain could be a good chassis for further biotechnological applications. HighlightsO_LICoelastrella sp. D14, a xero-tolerant strain, has been isolated from a solar panel C_LIO_LIThis strain can grow on piggery wastewater C_LIO_LICoelastrella sp. D14 can promote germination of Lepidium sativum C_LI

microbiology↗

bioRxiv2023

Unravelling the regulatory function of a short 3'UTR sequence in zebrafish and in human cells

The mechanisms of gene expression regulation are essential for cell identity and function, and their disruption usually leads to human disease. The 3 untranslated region (UTR) of mRNA contains important regulatory elements of gene expression, including upstream sequence elements (USEs) that are cis-regulatory sequences localized upstream of polyA signals (PAS). One of the best functionally characterised USEs is located in the 3UTR of the Drosophilas polo gene, which disruption leads to critical phenotypes in adult flies. In this work we found that the USE of the Drosophilas polo gene (DplUSE) is also found in in the 3UTR of vertebrate genes, including zebrafish, mouse and human genes, showing higher levels of conservation than the whole 3UTR sequence. Using reporter assays, we show that DplUSE is able to increase gene expression in vitro in human cell lines and in vivo in zebrafish embryos. Importantly, in humans, the DplUSE containing genes are enriched for genes associated to serious diseases such as Congenital abnormalities and Malignant neoplasms, illustrating the potential of this sequence to modulate genes with relevant biological functions and related with human health. Concomitantly, when sequestering the molecular machinery that operates at the DplUSE using a dominant negative strategy, we show that this is enough to dysregulate DplUSE containing genes in human cells and disrupt proper embryo development in zebrafish. Aiming to understand the molecular mechanism operating at the DplUSE, we identified three RNA binding proteins (RBP) that specifically bind to the DplUSE in vertebrates. Importantly, one of such RBPs is PTBP1, the vertebrate orthologue of the fruit flys RBP Heph, that was demonstrated to be required for the DplUSE function in Drosophila. To test if PTBP1is essential for the DplUSE function, as observed in Drosophila, we depleted PTBP1 from human cells and observed a downregulation of the expression of DplUSE containing genes, demonstrating that the molecular mechanisms that operate at DplUSE are ultra-conserved. Finally, we explored if variants in DplUSE consensus could be associated to human disease. We found a reported single nucleotide polymorphism (SNP; rs3087967) that is associated with malignant tumor of colon and generates an ectopic consensus of DplUSE in the 3 UTR of the tumorigenic POU2AF2/C11orf53 gene. We further show that this ectopic DplUSE motif causes a gain-of-function in vivo in zebrafish gut cells, suggesting its involvement in colon cancer development. These results show that a short motif present in the 3UTR of genes from phylogenetically distant bilaterians, from fruit flies to humans, control genes expression through an ultra-conserved mechanism involving RBPs binding and its dysregulation might impact in human disease.

molecular biology↗

bioRxiv2023

Delineation of the pathogenic presynaptic mechanisms of synaptotagmin-1 variants

Missense variants in the presynaptic calcium sensor synaptotagmin-1 (SYT1) are associated with an autosomal dominant neurodevelopmental disorder (NDD). These cause dominant-negative impairment of evoked neurotransmitter release in a mutation-specific manner; however, whether NDD-associated variants also perturb the auxiliary functions of SYT1 remains unknown. We investigated whether the expression of SYT1 variants in cultured hippocampal neurons altered either action potential-independent spontaneous synaptic vesicle exocytosis or synaptic vesicle endocytosis. SYT1 variants did not induce dominant-negative impairment of either process, confirming that defective evoked exocytosis is the major pathogenic mechanism of SYT1-associated NDD. To examine the differential impacts of human variants on evoked exocytosis, both NDD-associated and strategic mutations were used to explore the functional importance of two distinct Ca2+-binding residues in the C2B domain. We show that both the nature of the amino acid change and the specific residues targeted determine the severity of exocytic disturbance. Together, this work informs understanding of SYT1 function and further clarifies potential mechanistic targets for treating SYT1-associated NDD.

neuroscience↗

bioRxiv2023

Obesity-elicited macrophages shape CD9hi progenitor fate to promote adipose tissue fibrosis and dysfunction

Obesity is a life-threatening condition characterized by a maladaptive remodeling of the visceral white adipose tissue (vWAT), including fibrosis, that drives vWAT metabolic alterations. We previously identified CD9hi adipose tissue progenitors as the main drivers of vWAT fibrosis in mice and humans. However, how their functions are controlled, especially by macrophages, remains largely unknown. We found that obesity-elicited monocyte-derived macrophages (obeMac) accumulation was considerably elevated in mice prone to obesity-induced vWAT fibrosis. Limiting obeMac build-up decreased the numbers and fibrogenic activation of CD9hi progenitors, leading to decreased vWAT fibrosis and improved glucose homeostasis. In patients with obesity, we identified macrophages similar to mouse obeMacs that were associated with accumulation of CD9hi progenitors in the vWAT and loss of glycemic control. Finally, intercellular communication analysis identified mediators produced by obeMacs that control the fibrogenic potential of CD9hi progenitors. Together, we uncovered an obeMac-CD9hi progenitors axis controlling vWAT fibrosis and dysfunction.

pathology↗

bioRxiv2023

Shifting KRAS hotspot mutations inhibition paradigm in colorectal cancer

KRAS hotspot mutations are difficult to target, highlighting the need of developing new specific target drugs for cancers driven by these mutations, like colorectal cancer (CRC). Here, we discover a new ruthenium compound, PMC79, that inhibits specifically mutated KRAS and the downstream signaling ERK and AKT proteins both "in vitro" and "in vivo". We demonstrated that PMC79 inhibits KRAS mutated kinase activity and is selective for KRAS mutations not affecting the KRAS wild-type protein. KRAS inhibition is not dependent on actin polymerization or on proteasome. Molecular docking analysis suggests that this effect might result from protein dynamics associated with the mutations. We demonstrated that low doses of PMC79 potentiate 5-fluorouracil anticancer effect. "In vivo" PMC79 "proof of concept" showed that it reduces tumor growth in the CAM-xenograft model and induces necrosis of the tumor in the xenograft mice model. PMC79 is a promising new "magic bullet" for CRCs harboring mutated KRAS.

pharmacology and toxicology↗

bioRxiv2023

Distinct forms of structural plasticity of adult-born interneuron spines induced by different odor learning paradigms

During development and in adulthood the morpho-functional properties of neural networks constantly adapt in response to environmental stimuli and learned experiences. One of the processes that allows neuronal networks to be constantly reshaped is synaptic plasticity, which is induced in response to sensory experience and learning. Synaptic plasticity allows for the formation/elimination of synaptic connections as well as the strengthening of pre-existing ones. The olfactory system is particularly prone to constant morpho-functional reshaping of neural networks and synaptic rewiring throughout the lifespan of an animal, mainly because of the presence of continuous neurogenesis in the olfactory bulb (OB). This constant synaptic rewiring brought by adult-born neurons is modulated by the level of odor-induced activity and olfactory learning. It remains, however, unclear whether the complexity of distinct odor-induced learning paradigms and sensory stimulation induces different forms of structural plasticity. In the present study, we developed an analytical pipeline to perform 3D reconstructions of spines from confocal images followed by clustering of reconstructed spines based on different morphometric features and in relationship with different sensory stimuli and learning paradigms. We show that while sensory deprivation decreased the overall density of adult-born neurons in the OB without any noticeable changes in the morphometric properties of these spines, simple and complex odor learning paradigms triggered distinct forms of structural plasticity. A simple odor learning task affected the morphometric properties of the spines without any changes in spine density, whereas a complex odor learning task induced changes in spine density, without substantial changes in the morphology of the spines. Our work reveals the vast panoply of distinct forms of synaptic plasticity of adult-born neurons in the OB tailored to the complexity of odor-learning paradigms and sensory inputs.

neuroscience↗

bioRxiv2023

Integrative reappraisal of the Amazonian nurse frog Allobates gasconi (Morales 2002) based on topotypical data, with implications for the systematics and taxonomy of a large species complex

Taxonomic uncertainty at the species level compromises our knowledge of biodiversity, conservation and systematics. The impact of such uncertainty is heightened in megadiverse regions such as Amazonia due to high levels of cryptic diversity. We used integrative taxonomy based on newly collected topotypical specimens to redescribe the Amazonian nurse frog Allobates gasconi and infer its phylogenetic relationships. This species was described in 2002 based solely on morphology, but several characters crucial for the reliable diagnosis of species in Allobates were not considered. Our results show that A. gasconi sensu stricto is not a member of the A. caeruleodactylus clade as previously claimed, but is a member of the A. trilineatus clade. Allobates gasconi is readily distinguished from congeners by a combination of morphological and bioacoustic characters; a revised diagnosis is provided. The type series of A. gasconi comprises more than one species, and we exclude a paratype from lower Jurua River. The species is restricted geographically to flooded environments in the middle and upper Jurua River in Brazil and in the Ucayali River in Peru. The initial misidentification, subsequent absence of topotypic molecular and acoustic data, and the poor preservation condition of the type series have contributed to taxonomic confusion since A. gasconi was first described. The descriptions of other species of Allobates published more than two decades ago were based mainly on gross morphology and we recommend integrative taxonomic revisions to elucidate their systematics.

zoology↗

bioRxiv2023

Optimization of somatic embryogenesis in Euterpe edulis Martius using auxin analogs and atomic force microscopy

Euterpe edulis Martius is an endangered species of the Atlantic Forest, whose fruits have high antioxidant potential, and propagated exclusively by seeds. The present study assessed the ability of different auxin inducers and picloram analogs to trigger somatic embryogenesis in E. edulis. Immature seeds were harvested, and their zygotic embryos were excised and grown in MS culture medium supplemented with 2,4-D dichlorophenoxyacetic acid (2,4-D) or picloram at 150, 300, 450, 600 {micro}M. The activity of picloram analogs triclopyr and clopyralid was evaluated in semisolid MS medium. At maturation and germination, picloram-derived calli and somatic embryos isolated from triclopyr-grown cultures were first transferred to pre-maturation medium and, after 30 days, to basal MS or MS medium supplemented with either 5 {micro}M abscisic acid or 0.53 {micro}M 1-naphthaleneacetic acid plus 12.3 {micro}M 2-isopentenyladenine. Finally, somatic embryos with root protrusions were transferred to MS medium devoid of sucrose for 30 days and then acclimatized ex vitro. Scanning, transmission, and atomic force microscopy revealed that picloram was superior to 2,4-D but less effective than triclopyr (100 {micro}M) in inducing embryogenesis. Maturation and germination of somatic embryos in E. edulis can be maximized by 5 {micro}M abscisic acid, and selecting calli via atomic force microscopy. HighlightThis work opens novel roads for embryogenic induction, using a new and more efficient inducer than the usual ones, and an innovative evaluation technique based on AFM.

plant biology↗

bioRxiv2023

A novel cryopreservation and biobanking strategy to study lymphoid tissue stromal cells in human disease

Non-hematopoietic lymph node stromal cells (LNSCs) regulate lymphocyte trafficking, survival, and function for key roles in host defense, autoimmunity, alloimmunity, and lymphoproliferative disorders. However, study of LNSCs in human diseases is complicated by a dependence on viable lymphoid tissues, which are most often excised prior to establishment of a specific diagnosis. Here, we demonstrate that cryopreservation can be used to bank lymphoid tissue for the study of LNSCs in human disease. Using human tonsils, lymphoid tissue fragments were cryopreserved for subsequent enzymatic digestion and recovery of viable non-hematopoietic cells. Flow cytometry and single-cell transcriptomics identified comparable proportions of LNSC cell types in fresh and cryopreserved tissue. Moreover, cryopreservation had little effect on transcriptional profiles, which showed significant overlap between tonsils and lymph nodes. The presence and spatial distribution of transcriptionally defined cell types was confirmed by in situ analyses. Our broadly applicable approach promises to greatly enable research into the roles of LNSC in human disease.

immunology↗

bioRxiv2022

Targeting the BAG-1 family of co-chaperones in lethal prostate cancer.

Therapies that abrogate persistent androgen receptor (AR) signaling in castration resistant prostate cancer (CRPC) remain an unmet clinical need. The N-terminal domain (NTD) of the AR drives transcriptional activity in CRPC but is intrinsically disordered and remains a challenging therapeutic target. Therefore, inhibiting critical co-chaperones, such as BAG-1L, is an attractive alternative strategy. We performed druggability analyses demonstrating the BAG domain to be a challenging drug target. Thio-2, a tool compound, has been reported to bind the BAG domain of BAG-1L and inhibit BAG-1L-mediated AR transactivation. However, despite these data, the mechanism of action of Thio-2 is poorly understood and the BAG domain which is present in all BAG-1 isoforms has not been validated as a therapeutic target. Herein, we demonstrate growth inhibiting activity of Thio-2 in CRPC cell lines and patient derived models with decreased AR genomic binding and AR signaling independent of BAG-1 isoform function. Furthermore, genomic abrogation of BAG-1 isoforms did not recapitulate the described Thio-2 phenotype, and NMR studies suggest that Thio-2 may bind the AR NTD, uncovering a potential alternative mechanism of action, although in the context of low compound solubility. Furthermore, BAG-1 isoform knockout mice are viable and fertile, in contrast to previous studies, and when crossed with prostate cancer mouse models, BAG-1 deletion does not significantly impact prostate cancer development and growth. Overall, these data demonstrate that Thio-2 inhibits AR signaling and growth in CRPC independent of BAG-1 isoforms, and unlike previous studies of the activated AR, therapeutic targeting of the BAG domain requires further validation before being considered a therapeutic strategy for the treatment of CRPC.

molecular biology↗